Individualized Reconstitution of Human Milk Microbiota: A Feasible Approach in Real-World Settings

We are investigating whether it's possible to recover the microbiota of a specific mother's milk preserving nutritional structure in pasteurized milk through methodology that can be implemented in hospitals. Our goal is to develop a new nutritional super strategy for newborns and assess its visibility. We believe that the greatest challenge in reconstituting the human milk microbiota experimentally is achieving a reconstruction that is even closer to the original profile present in the mother's milk, and doing so quickly.

This will not only enhance its effects, but also further increase the safety of the procedure. We aim to determine whether these new nutritional super strategy offers significant clinical benefits for neonates. We believe that if practical benefits are confirmed, the strategy could be easily implemented in all regions with human milk banks leading to a positive impact on overall non-adult health.

After obtaining the required consent, have the mother express milk using an electric breast pump into a sterilized container under the supervision of the nurse. After collection, label the container holding the milk with the mother's information, the date, and the time of collection. Store the mother's own milk or MOM at minus four degrees Celsius or lower for up to 12 hours.

Thaw raw milk or pasteurized donor milk or PDM, stored in sterilized glass containers with plastic lids in a 40 degree Celsius water bath. Remove the human milk bottle from the water bath before the milk temperature exceeds five degrees Celsius. Next, wear a lab coat, mask, cap, and gloves.

Clean the work surface with a surface cleaning solution, following the local institutional protocol. Transfer the aliquot of up to one milliliter of the MOM into a cryotube for subsequent 16S sequencing and bacterial count. Store the cryotube at minus 80 degrees Celsius.

Using a sterile pipette, transfer the remaining volume of MOM, equivalent to 10%of the final desired volume, to a food safe container. Immerse the container in a water bath at 37 degrees Celsius. Next, transfer the one milliliter aliquot of the PDM into a DNase/RNase Free cryotube for 16S sequencing and bacterial count.

Store the cryotube at minus 80 degrees Celsius. Using a sterile pipette, transfer an amount of PDM equivalent to 90%of the final desired volume to the container holding the MOM, forming the inoculated mother's milk. Seal the container with a lid to prevent contamination and immerse it in a 37 degree Celsius water bath for four hours to allow microorganisms to multiply in the milk.

After four hours, remove the container with the fermented inoculated mother's milk from the water bath. Transfer the one milliliter aliquot of the reconstituted milk into a DNase/RNase Free cryotube for 16S sequencing and bacterial count. Store the cryotube at minus 80 degrees Celsius.

Transfer the remaining volume of reconstituted milk to a sanitized container for feeding newborns.